Determination of isoflavones in dietary supplements containing soy, Red Clover and kudzu: Extraction followed by basic or acid hydrolysis

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Abstract

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to β-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Dietary supplement preparations contain extracts from soy, Red Clover and kudzu. Soy products contain primarily genistein, daidzein, and glycitein, while Red Clover products contain primarily formononetin and biochanin A. Kudzu extracts contain puerarin and daidzein among other components. Previous methods of analysis focused on the determination of isoflavones from a single botanical source, while dietary supplements are often a blend of extracts from different plants. We developed a method for the analysis of isoflavones in dietary supplements regardless of their botanical composition, using HPLC-PDA because of its applicability to routine analysis. Isoflavones are found as free compounds, glucoside derivatives, 6″-O-malonyl-β-d-glucoside and 6″-O-acetyl-β-d-glucoside derivatives. In this study, the samples were extracted at room temperature with 50:50 (v/v) MeCN/water, and then analyzed before and after hydrolyzing the isoflavones by acid or basic digestion. 2′-Methoxy-flavone and 6-methoxy-flavone were used as internal standards and were added together to every sample. Daidzein, glycitein, genistein, puerarin, calycosin, pratensein, pseudobaptigenin, formononetin, biochanin A and prunetin were among the isoflavones determined.

Introduction

Isoflavones are compounds with estrogenic activity (phytoestrogens) that are widely distributed in the seeds and other parts of many plant species belonging to the Leguminosae family. Soybeans, alfalfa, clover and chickpeas have all been found to contain a range of phytoestrogens [1]. Because of their structural similarity to β-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases [1], [2]. Isoflavones are characterized by a common 3-phenyl-chromen-4-one core structure, and differ by substituents such as methoxy, hydroxyl and glycoside functions on the primary structure. Fig. 1 shows the structure of the main isoflavones that have been reported in food and that are commercially available as reference materials. Isoflavones are usually found in processed foods as free compounds and 7-O-β-d-glucoside derivatives. In plants, the 6″-O-malonyl-7-O-β-d-glucoside derivatives are the predominant forms [3], [4], [5], [6], [7] and 6″-O-acetyl-7-O-β-d-glucosides are produced from them (along with 7-O-β-d-glucosides and free isoflavones) during food processing or sample preparation [5], [8], [9], [10].

Isoflavone dietary supplements generally contain extracts from soy, Red Clover and kudzu plants. Each one of these plants presents a characteristic isoflavone profile. Soybeans and soy foods contain primarily daidzein, glycitein and genistein, in the form of aglucones, glucosides, malonyl-β-glucosides and acetyl-β-glucosides according to the food processing and sample preparation [5], [8], [9], [10], [11]. Formononetin and biochanin A, present as 6″-O-malonyl-7-O-β-d-glucosides, 7-O-β-d-glucosides and aglucones, are reported as the most abundant isoflavones in Red Clover [12], [13]. Wu et al. [14] identified by LC-MS/MS 10 different isoflavones (daidzein, genistein, glycitein, formononetin, pseudobaptigenin, calycosin, prunetin, biochanin A, irilone and pratensein), and a total of 31 derivatives, in an extract of Trifolium pratense leaves. Pueraria lobata (Willd.) Ohwi (common name “kudzu”), a perennial vine native to Japan and China that also grows in the Southeastern portion of the U.S., is a commercial source of isoflavones for dietary supplements. The isoflavone content of kudzu is quite complex and seems to have been less studied than those of soy and Red Clover. Puerarin (daidzein 7-C-β-d-glucoside), daidzin (daidzein 7-O-β-d-glucoside) and daidzein are the main isoflavones identified in a methanolic extract of Pueraria radix [15]. Isoflavone di-glucosides such as daidzin-4′-O-glucoside and puerarin-4′-O-glucoside, along with other unusual isoflavones, were also identified in kudzu extracts by LC-MS/MS analysis [16].

Extraction of isoflavones from foods or dietary supplements is a critical process since the composition of isoflavone mixtures (free or derivatives) can be altered during sample preparation. Earlier methods have utilized refluxing alcohol, which results in the conversion of the 6″-O-malonyl-glucosides and 6″-O-acetyl-glucosides to glucoside and aglucone forms [17], [18]. Recent methods have used room temperature or chilled extractions, in order to decrease the degradation of isoflavone derivatives [10]. An acid or basic hydrolysis step is often included in the sample preparation in order to hydrolyze isoflavone derivatives to simpler forms [13], [19]. Based on the results presented by Griffith and Collison [5], the MeCN/water at room temperature procedure was preferred for this methodology.

The experimental conditions of previous studies were optimized for the identification and quantification of isoflavones from a single botanical source, while dietary supplements currently available on the market are often a blend of extracts from different plants. The purpose of this study was to develop a methodology capable of quantifying isoflavones in dietary supplements regardless of the botanical composition. Samples were analyzed directly after extraction, and after acid or basic hydrolysis of isoflavone derivatives to simpler forms. Puerarin, daidzein, glycitein, genistein, calycosin, pratensein, pseudobaptigenin, formononetin, biochanin A and prunetin were the isoflavones considered in this study. Daidzein, glycitein and genistein were also investigated as 7-O-β-glucosides (daidzin, glycitin and genistin, respectively), 6″-O-malonyl-7-O-β-glucosides and 6″-O-acetyl-7-O-β-glucosides. Formononetin and biochanin A were also investigated as their 7-O-β-glucosides.

Section snippets

Reagents

Acetonitrile, dimethylsulfoxide (DMSO), acetic acid, hydrochloric acid and ammonium hydroxide were purchased from J.T. Baker (Phillipsburg, NJ, USA). Isoflavone standards were obtained from several suppliers. Daidzein, daidzin, 6″-O-acetyl-daidzin, 6″-O-malonyl-daidzin 6″-O-acetyl-glycitin, 6″-O-malonyl-glycitin, genistein, genistin, 6″-O-acetyl-genistin and 6″-O-malonyl-genistin were purchased from LC Laboratories (Woburn, MA, USA). Glycitein, glycitin, formononetin, formononetin-7-O-β-d

Internal standards

Several internal standards have been used for the analysis of isoflavones from known sources. Apigenin (the flavone analog of genistein) and fluorescein [5] have been used in a number of studies of isoflavones in soy-containing samples. Barnes et al. [21] suggested the use of apigenin for LC-MS analysis of isoflavones, but were not able to resolve apigenin from genistein. However, genistein and apigenin are well resolved in the gradient HPLC method reported by Griffith and Collison [5]. In the

Conclusions

The 90 min elution with a MeCN/water gradient allowed the separation of almost all of the isoflavones commercially available, allowing the simultaneous quantification of isoflavones (free or as derivatives) present in soy, Red Clover and kudzu based dietary supplements. For the samples considered in this study, primarily soy, Red Clover and kudzu extracts, 2′-methoxy-flavone and 6-methoxy-flavone were considered suitable internal standards, and were quantitatively recovered after acid or basic

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